Periodontitis is an inflammatory disease triggered by host immune response to pathogenic microorganisms in the periodontal biofilm. B lymphocytes are densely infiltrated at the site of infection and are a primary source of receptor activator of NF-:B ligand (RANKL), a cytokine that plays a pivotal role in osteoclast differentiation and inflammatory bone resorption. Our long-term goal is to determine the molecular mechanisms which control immune cell-mediated periodontal bone resorption in periodontitis for preventive and therapeutic purposes. Toll-like receptors (TLRs) recognize pathogen associated molecular patterns (PAMPs) and TLR signaling pathways play an important role in regulating immune cell functions, including cytokine production, phagocytosis, and programmed cell death (apoptosis). While various B lymphocyte subsets express multiple TLRs, including TLR4 and TLR9, the role of TLR signaling on B cell apoptosis is entirely unclear. The central hypothesis of this application is that co-activation of TLR4/TLR9 signaling induces apoptosis of RANKL-producing B cells, thus diminishing B cell-mediated periodontal bone resorption. Our hypothesis is based on our preliminary results demonstrating that co-stimulation with TLR4/9 agonists, LPS and CpG ODN, elevated expressions of TLR4 and TLR9 and increased B cell apoptosis in cultured rat splenocytes. In this application we will explore how TLR4/TLR9 signaling regulates B cell apoptosis using both in vitro and in vivo models. In Specific Aim 1, we will determine if co-activation of TLR4 and TLR9 induce RANKL-expressing B cell apoptosis, thereby inhibiting B cell-mediated osteoclastogenesis, using an in vitro osteoclastogenesis model. In Specific Aim 2, we will determine whether co-activation of TLR4/TLR9 induces B cell apoptosis and decreases B cell-mediated periodontal bone resorption in vivo. We have developed an in vivo model of adoptive B cell transfer/gingival antigen injection that can be used to undertake the proposed research. The rationale for the proposed research is that, once it is known how TLR signaling controls RANKL-expressing B cell apoptosis, therapeutic strategies based on induction of RANKL-expressing B cell apoptosis, therefore, inhibition of B cell-mediated osteoclastogenesis may be effective in preventing bone resorption and tooth loss in people with periodontitis. It is also expected that what is learned here could be applicable to the understanding of other immune-mediated bone-resorptive disorders.